We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). The factor eIF2 is composed of three polypeptide chains. The gamma subunit of eIF2 is a GTPase that, based on sequence and the structure of the archaeal homolog aIF2gamma, resembles elongation factor EF-Tu. However, in contrast to EF-Tu, which binds tRNAs to the A-site of the 70S ribosome, eIF2 binds Met-tRNAi to the P-site of the 40S subunit. To gain insights into how eIF2 binds Met-tRNAi and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40S-eIF1-eIF1A-eIF2-GTP-Met-tRNAi-mRNA (48S) complex. Based on the results of our experiments, and the fact that Met-tRNAi is bound to the P-site of the 40S subunit, we generated a model of the 48S complex in which domain III of eIF2gamma binds near 18S rRNA helix h44 and eIF2gammaMet-tRNAi contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAi is rotated nearly 180 degrees relative to the position of the tRNA in the EF-Tu ternary complex. Consistent with the alternate models of the eIF2 and EF-Tu ternary complexes, we found that a domain III mutation in EF-Tu severely impaired Phe-tRNA binding; whereas, the corresponding eIF2gamma mutation did not impair Met-tRNAi binding to eIF2. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNA in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. Whereas protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Moreover, the consequences of partial loss of eIF2gamma function or eIF2 integrity are unknown in mammals, including humans. Our collaborators in Israel and Germany identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Thus, a hypomorphic genetic alteration in the eIF2gamma gene causes an intellectual disability syndrome in humans. Biochemical studies of human cells overexpressing the eIF2gamma mutant and of yeast eIF2gamma with the analogous mutation revealed a defect in binding the eIF2beta subunit to eIF2gamma. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2gamma impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2beta. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. Previously, we demonstrated that, when expressed in yeast, human PKR phosphorylates the alpha subunit of eIF2 on Ser51 resulting in inhibition of protein synthesis and yeast cell growth. We also identified the mechanism of activation of PKR that requires back-to-back dimerization of two PKR kinase domains. In order to subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively, restored yeast cell growth. We are currently characterizing mutations in both the PKR kinase domain and its regulatory domain that confer resistance to E3L inhibition. As the kinase domain mutations enhance PKR dimerization, these results suggest that E3L may inhibit PKR by blocking kinase dimerization. The E3L protein consists of an N-terminal Z-DNA binding domain (ZBD) and a C-terminal double-stranded RNA (dsRNA) binding domain (RBD). Whereas both E3L domains are required for viral pathogenicity, the function of the ZBD has not been resolved. Our preliminary data indicate that whereas the RBD is absolutely required for E3L inhibition of PKR, the requirement for the ZBD is dependent on the assay system. Thus, both the ZBD and RBD are required for PKR inhibition in yeast cells; however, the ZBD is non-essential for inhibition of PKR in typical mammalian cell assays. Interestingly, whereas the ZBD is required for E3L inhibition of PKR in yeast, mutations designed to disrupt nucleic acid binding to the ZBD do not affect PKR inhibition. These results suggest that the ZBD of E3L has additional functions beyond nucleic acid binding. We are also studying the translation factor eIF5A. eIF5A is the sole protein containing the unusual amino acid hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine. This hypusine residue in eIF5A is found in all eukaryotes and archaea. The eIF5A was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A was unknown. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we proposed that eIF5A/EF-P is a universally conserved translation elongation factor. Recently, it was shown that EF-P promotes translation of polyproline sequences by bacterial ribosomes. Using in vivo reporter assays, we show that eIF5A in yeast stimulates the synthesis of proteins containing consecutive proline residues. Consistently, the expression of native yeast proteins containing homopolyproline sequences is impaired in eIF5A mutant strains. Consistent with these in vivo findings, synthesis of polyproline peptides, but not polyphenylalanine peptides, in reconstituted yeast in vitro translation assays is critically dependent on addition of eIF5A. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive proline residues in vitro. Consistent with these functions of eIF5A in promoting peptide bond synthesis, directed hydroxyl radical probing experiments localized eIF5A binding near the E site of the ribosome with the hypusine residue of eIF5A adjacent to the acceptor stem of the P-site tRNA. Thus, we propose that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosomes and facilitates the reactivity of poor substrates like proline.